The synthesis of ATP during oxidAtive phosphorylation is catalyzed by a reversible H+-translocating ATP synthase complex. The H+ ATP synthase, or H+ ATPase, is composed of two sectors: F1 is the ATPase moiety and is easily dissociated from the membrane, whereas F0 extends through the membrane and functions as the H+- translocase. The long term challenge before us is to define how the F0 moiety translocates H+, and how H+ flux through F0F1 is coupled to ATP synthesis. In Escherichia coli, F0 is composed of 3 types of subunits (a, b, and c), and F1 of 5 types of subunits. These subunits are coded by the unc operon which has been cloned and sequenced. The F1F0 complexes of E. coli and mitochondria are very similar, so conclusions from one should be applicable to the other. This grant is aimed at providing information on the functional role of subunits c and a through analysis of mutants, as well as more information on structure. Subunit c is small (79 amino acids) and will be analyzed in greatest detail. We will determine the regions and specific residues that are essential to function by saturation mutagenesis, the optimal position and critical interactions of an essential carboxyl by moving it around, and regions of F1 that are "coupled" with subunit c by analysis of second site revertants. Subunit c is predicted to fold like a hairpin in the membrane, and must also do so in organic solvent as judged by 1H NMR, and retention of inhibitor binding properties. The structural and functional properties is organic solvent will be correlated with the properties in fully assembled F0, in wild-type and mutant membranes, and mutant proteins will be examined for predicted changes in structure. Functional domains of the larger subunit a will be defined by random mutagenesis, and interesting hot spot areas further characterized by localized mutagenesis. The folding of subunit a in the membrane will be evaluated using antibodies directed against putatively external peptide sequences. The combined genetic and NMR structural approaches with subunit c potentially could provide a high resolution picture of the central component of a membrane H+-translocator. These studies should therefore provide insight into no only the mechanism of oxidative phosphorylation, but also of membrane transport. A detailed understanding of membrane transport proteins, ATPase-pumps, etc. is fundamental to many areas of medical science, including the pathology of many diseases.